Dynamic stress microenvironments can modulate the biomechanics of cells resulting in distinctly different signatures for normal and caner cells. The proposed research aims at analyzing the biophysical changes occurring in breast cells when they are excited under repetitive forces. In this proposal, we plan to expose cells to sequential deformations and to identify a more comprehensive biomechanical marker for cancer diagnosis, prognosis, and treatment. The proposed ?mechanical modulatory signatures? result from changes in the cell velocity as it traverses through multiple constriction regions and can hypothetically predict the metastatic potential and drug responsiveness of breast cancer cells. Our previous work with atomic force microscopy and microfluidic chips reveal that breast cancer cells are softer and more fluidic than their healthy counterparts. Moreover, cancer cells demonstrate strain-softening behavior while normal cells display strain-stiffening or less softening attitude. Our research outcome will have substantial impact on breast cancer biology and drug development as it implies that cancer cells as they leave their original site can become softer by squeezing through pores to reach to blood vessels and metastasize while normal cells show more resistance and hence their migration slows down and potentially stops. Aim 1 is to develop a high throughput microfluidic chip and the corresponding fluidic and image processing interfaces to analyze the mechanical modulatory signature of single cells as they pass through multiple constrictions. Both normal and cancer cell lines and primary cells will be used. Different constriction architectures will be explored by varying the overall channel length and the relaxation regions between two subsequent constriction regions. Upon successful accomplishment of this phase of the project, we will realize a high throughput assay enabling the biomechanical analysis of about 50,000 cells per minute. The bioassays will be used to discover if there are unique modulatory signatures for each enlisted cell category (non-invasive, moderately invasive, and highly-invasive) that can be used to distinguish them and how these signatures are related to the constriction architecture. Aim 2 will be to assess the role of chemotherapy agents on cell biomechanical signatures and their corresponding cytoskeletal architectures. Aim 2 is a fundamental study defining the impact of microtubulin disrupting drugs on the mechanics of living breast cells. Both anti-cancer microtubulin stabilizer and destabilizer drugs will be used and their effect on biomechanical modulatory signatures of cell lines and primary cells will be determined. This aim will identify if cell mechanical signatures have changed upon drug treatment and if cell softening/stiffening observed due to cyclic deformations have altered and to which degree.